Diagnostic assays play a significant role in the detection and diagnosis of diseases. The development of diagnostic assay technology has made possible the precise measurement of minute amounts of analyte in samples of serum, plasma, saliva, cerebral spinal fluid, amniotic fluid and urine. Applications of such assays include measuring drug concentrations administered to patients for the treatment of diseases and detecting blood components resulting from diseases including cancer. Thus, their applications in the fields of biology and medicine have made them increasingly important and versatile as diagnostic tools.
There are complications, however, with the uses of assays. The sensitivity and accuracy of these assays are often limited by the instability of the analyte of interest due to the conditions of the assay. Such limitations can make detection and measurement of the analyte of interest unreliable, as well as decrease the sensitivity of the assay.
The effects of these limitations can be disastrous since their presence in assays could cause inaccurate results leading to misdiagnosis and potentially inappropriate treatment.
In certain liver diseases such as hepatitis, liver cirrhosis, or obstructive jaundice, the diconjugate bilirubin species is believed to be elevated in early stages of the disease.
Bilirubin is a typical metabolic product of hemoglobin which is an oxygen carrier in blood. Determination of the amount of bilirubin in body fluid, especially blood, is important for detection of hemolysis and for checking liver function. One symptom of excess bilirubin in blood is jaundice. Determination of the bilirubin content in blood has been an important item of clinical chemical testing.
Bilirubin is a yellow colorant which itself has an absorption peak at 435 nm or 465 nm and molecular absorptivity coefficient of about 5.times.10.sup.4 C.sub.33 H.sub.36 O.sub.6 N.sub.4.
Bilirubin can be detected by examining the density of green color formed when bilirubin is oxidized by an oxidizer to biliverdin and by knowing precisely how this density is proportional to bilirubin content. This method is associated with qualitative testing. Disadvantages with this method is that it can be difficult to quantitate the amount of bilirubin in a sample.
Various calorimetric methods for determining bilirubin concentration have been proposed. In one method, bilirubin is coupled with a diazonium salt such as diazosulfanilic acid and the amount of the resulting colorant is measured in a spectrophotometer to estimate the bilirubin content. However, since the bilirubin content in the blood serum of a healthy subject is very small, it can be difficult to achieve accurate determination of the bilirubin concentration using this method. bilirubin is extracted from an aqueous solution with a hydrophobic organic solvent. A disadvantage with this method is that it can take time to extract the bilirubin from an aqueous solution and it can require large amounts of sample which can be difficult to concentrate, which can lead to low extraction recovery. A further disadvantage associated with this method is that this method may not be specific for measuring the conjugated species of bilirubin.
For the foregoing reasons, there is a need for a sensitive specific assay that can accurately determine the presence of an analyte in a sample that undergoes auto-oxidation. Further, it would be advantageous to have an assay which would be capable of satisfying the above criteria yet able to maintain the standardization of the assay by avoiding auto-oxidation.